The invention relates to a tissue adhesive which contains fibrinogen, factor XIII, a thrombin inhibitor, prothrombin factors, calcium ions and, where appropriate, a plasmin inhibitor. The accelerators which are naturally present on the wound which is to be bonded result in the thrombin which is necessary for adhesion being liberated from the prothrombin in the adhesive.
The physiological processes involved in the repair of lesions in human or animal tissues result in fibrin being deposited in the region of the injury. This is initiated by thromboplastin flowing out of the injured cells and, on contact with plasma factor VII forming factor X activator which then, together with factor V and in a complex with phospholipids and calcium, converts prothrombin into thrombin. The action of thrombin in turn results, via elimination of the fibrinopeptides A and B from fibrinogen, in fibrin monomers which aggregate to form fibrin filaments which are covalently cross-linked, with the formation of isopeptide bonds by F XIIIa, a transglutaminase which is likewise activated by thrombin. An inhibitor, namely alpha2-antiplasmin, is bound by factor XIII onto the fibrin filaments and protects them from degradation by plasmin.
It is evident from this process of hemostasis and wound healing that, in addition to fibrinogen as substrate, two enzymes are required: thrombin and factor XIII.
The task of thrombin in this connection is to activate both fibrinogen and factor XIII, that is to say to convert them into their reactive form. This is the fibrin monomer in the case of fibrinogen, and is the catalytically active transglutaminase (F XIIIa) in the case of factor XIII.
European Patent 0,068,047 discloses an enriched plasma fraction which is suitable as a wound closure and contains fibrinogen, a fibrinolysis inhibitor and thrombin or prothrombin in an anhydrous system. This adhesive cannot be applied dissolved in water, but can be applied only as a powder, which is a disadvantage for use. When the mixture is dissolved in water, the components react together, and a clot is produced.
For a one-component adhesive with water as solvent it is difficult to combine the active substances in such a way and in such quantitative ratios that no premature and undesired activation takes place during the production of the adhesive, the reconstitution with a solvent and the maintenance in readiness for use and, on the other hand, a sufficiently high strength is achieved relatively quickly after application to the adhesion site.
Surprisingly, conditions allowing reproducible production of a one-component adhesive, which can be manipulated in practice, in a physiological medium have been found.
The requirements for a one-component adhesive of this type to be produced and be amenable to processing are that the relative concentrations of fibrinogen, plasmin inhibitor, F XIII, prothrombin concentrate, thrombin inhibitor and calcium ions are optimized. This particularly applies to AT III as thrombin inhibitor and to calcium ions, since the calcium ions are essential for activation of the coagulation factors. High concentrations increase the rate of thrombin formation, and Low concentrations slow it down. Hence it is necessary that the concentration of calcium ions is optimal. Since, when this is the case, the formation of thrombin cannot be ruled out, it is expedient to combine with a thrombin inhibitor, preferably AT III. This is because, on the one hand, the formation of thrombin must be ruled out during the production of the adhesive and, on the other hand, the adhesive must rapidly form a stable clot when coagulation, that is to say the deposition of a fibrin film, is initiated on contact with wound surfaces. AccordingLy, the production of a thrombin-containing one-component adhesive and its use in aqueous solution is inconceivable and, moreover, it is possible to use prothrombin only under conditions which prevent activation to thrombin during production and preparation for use. These include setting up defined concentrations of thrombin inhibitor and calcium ions.
Thus the invention relates to a tissue adhesive containing in aqueous solution fibrinogen, F XIII, a thrombin inhibitor, prothrombin factors, calcium ions and, where appropriate, a plasmin inhibitor.
It was surprising that it is possible to pack, and use as tissue adhesive, fibrinogen together with prothrombin as a so-called hypercoagulable solution which, moreover, also contains calcium ions and AT III.
An adhesive of this type according to the invention can be converted into a solid form, for example freeze-dried, and reconstituted with water. It can contain all the active substances in pasteurized form, and is then free of the risk of transmission of hepatitis and HTLV III.
The mixing ratio of the active substances fibrinogen, plasmin inhibitor, F XIII, prothrombin factors, calcium ions and AT III is such that no premature activation takes place and such that, however, the mixture spontaneously coagulates, and deposits fibrin, on contact with wound surfaces.
The time in which this takes place is consistent with clinical requirements. The one-component adhesive according to the invention does not differ in its action from the two-component systems of the state of the art.
The mixture of active substances in this adhesive is such that, in the solution for use, the concentration of fibrinogen is 70-90 mg/ml and that of the prothrombin factors is between 10 and 30 U/ml based on F II. The concentration of calcium ions, some of which are protein-bound in the adhesive, is intended to be 0.5-1 mmol/l in the solution for use. Examples of thrombin inhibitors are AT III, hirudin and heparin, and AT III is preferably used in a concentration of 0.1-1 U/ml of tissue adhesive; at this concentration it prevents, at the abovementioned concentrations of prothrombin factors and calcium ions, premature fibrin formation. On the other hand, however, it is possible to favor the latter when this appears desirable on the basis of the indication. For this purpose, it is possible to dissolve the freeze-dried adhesive in a higher final concentration of calcium ions than 0.5-1 mmol/l. However, rapid use after reconstitution is then necessary.
To produce the adhesive, the active substances are initially produced in soluble form having the highest possible activity and, where appropriate, having been pasteurized. The fibrinogen concentration should be as high as possible, since the strength of adhesion increases with increasing concentration. The fibrinogen can, for example, be produced and pasteurized as described in European Patent 0,103,196, and given the desired solubility by additives of the type of compound containing urea or guanidine residues as described in European Patent 0,085,923. Factor XIII can be pasteurized as described in European Patent 0,018,561, and the prothrombin factors can be pasteurized as described in European Patent 0,056,629.
The active substances in the preferred adhesive according to the invention in principle comprise human fibrinogen in the highest possible concentration, enriched with factor XIII and the factors of the prothrombin complex (factor II, VII, IX and X), calcium ions and antithrombin III. The tissue adhesive also contains an inhibitor which inhibits plasminogen activators and/or plasmin and thus protects the resulting fibrin from degradation by plasmin. This inhibitor may be in the form of a concomitant substance of the fibrinogen selected for the bonding, but is preferably added to the adhesive in the form of aprotinin. To improve the reconstitution of the tissue adhesive after freeze-drying, it is possible for the latter to contain albumin and/or substances which contain the urea or guanidine residue and also contain, where appropriate, an amino acid with a hydrophobic side-chain or a water-soluble fatty acid and, in addition, heparin.
The fibrinogen which is suitable and preferred for preparation of the tissue adhesive is purified and already contains factor XIII. However, apart from this, also suitable is cryoprecipitate which, experience has shown, contains alpha2-antiplasmin and human albumin in addition to factor XIII. It is also possible, however, to produce an adhesive of comparable action by mixing the individual components, namely: human fibrinogen, factor XIII (from plasma or placenta), plasmin inhibitors, prothrombin factors (factor II, VII, IX and X), albumin, AT III and calcium ions, a compound with a urea or guanidine residue, or an amino acid with a hydrophobic side-chain or a water-soluble fatty acid.
The mixture of the active compounds for the adhesive is advantageously in the form of a solid, preferably a lyophilizate.
The tissue adhesive preferably contains the following quantities of active compounds, which are pasteurized where appropriate, based on 1 ml of solution for use:
65-115, preferably 70-90, mg of highly purified human fibrinogen
40-80 U of factor XIII
1-50, preferably 10-30, IU of PPSB (prothrombin factors) based on F II (prothrombin)
0-10,000 KIU of aprotinin
0.01-50, preferably 0.1-1, IU of antithrombin III
0-5 USP U heparin and
0.1-5, preferably 0.5-1 mmol/lofcalcium ions, preferably as calcium chloride.
The adhesive can contain, for example glutamate, L-iso-leucine, L-arginine or human albumin as stabilizers and solubilizers.
The mixture of the solid active compounds of the tissue adhesive can be dissolved in water, or in a solvent containing calcium ions, in a short time at 20xc2x0 C. When applied to the tissue which is to be united or bonded the adhesive develops within a sufficiently short time a high bonding strength without thrombin being necessary as a second component.
Accordingly, it is possible to apply the adhesive in liquid form using a single syringe, so that preparation, manipulation and use are straightforward. The adhesive is tolerated and is completely absorbed.
This one-component adhesive does not differ, either in the bonding time or in the rupture strength, from a two-component adhesive, as has been found in bonding tests in the model of puncture wounds of the rat skin and in the bonding of small intestinal anastomoses in hogs. It is even possible to increase the bonding strength by choosing an optimal calcium ion concentration of the solution which is used to dissolve the freeze-dried active compounds.
The examples which follow are intended to illustrate the invention.